The invention relates to novel cascade polymer complexing compounds and complexes, agents containing these compounds, the use of the complexes in diagnostics and therapy, as well as processes for the production of these compounds and agents.
xe2x80x9cMagnevistxe2x80x9d (GdDTPA/dimeglumine) is the first recorded contrast medium for nuclear spin tomography (MRI=magnetic resonance imaging). It is particularly well suited for the diagnosis of pathological areas (e.g., inflammations, tumors, etc.). The compound is eliminated, upon intravenous injection, by way of the kidneys; extrarenal elimination is practically not at all observed.
One disadvantage of xe2x80x9cMagnevistxe2x80x9d resides in that it is distributed after intravenous administration uniformly between the vasal and interstitial spaces. Accordingly, contrasting of the vessels with respect to the surrounding interstitial space is impossible with the use of xe2x80x9cMagnevistxe2x80x9d.
Especially for the imaging of vessels, a contrast medium would be desirable which is distributed exclusively in the vasal space (vascular space). Such a blood pool agent is to make it possible, with the aid of nuclear spin tomography, to demarcate tissue with good circulation from tissue with poor circulation, and thus to diagnose an ischemia. Also infarcted tissue could be distinguished, on account of its anemia, from surrounding healthy or ischemic tissue with the use of a vasal contrast medium. This is of special importance in case the objective is, for example, to distinguish a cardiac infarction from an ischemia.
Heretofore, most of those patients suspected of harboring a cardiovascular disease (this disease being the most frequent cause of death in Western industrial countries) had to undergo invasive diagnostic tests. In angiography, X-ray diagnostics is presently used, above all, with the aid of iodine-containing contrast media. These tests are burdened by various drawbacks: they bring the risk of radiation stress, as well as discomfort and strain stemming, above all, from the fact that the iodine-containing contrast media must be utilized in a very much higher concentration as compared with NMR contrast media.
Therefore, there is a need for NMR contrast media which can mark the vasal space (blood pool agent). These compounds are to be distinguished by good compatibility and by high efficacy (great increase in signal intensity during MRI).
The premise of solving at least part of these problems by the use of complexing agents bound to macro- or biomolecules has thus far been successful to only a very limited extent.
Thus, for example, the number of paramagnetic centers in the complexes described in European Patent Applications No. 88,695 and No. 150,884 is inadequate for satisfactory imaging.
When increasing the number of required metal ions by repeated introduction of complexing units into a macromolecule, the result is an intolerable impairment of the affinity and/or specificity of this macromolecule [J. Nucl. Med. 24:1158 (1983)].
Macromolecules are generally suited as contrast media for angiography. Albumin-GdDTPA (Radiology 1987; 162:205), for example, shows, however, an accumulation in liver tissue to an extent of almost 30% of the dose 24 hours after intravenous injection in rats. Besides, only 20% of the dose is eliminated within 24 hours.
The macromolecule polylysine-GdDTPA (European Patent Application, Publication No. 0,233,619) likewise proved to be suitable as a blood pool agent. However, this compound, on account of its production, consists of a mixture of molecules of various sizes. In elimination tests on rats, it could be demonstrated that this macromolecule is eliminated unchanged by glomerular filtration via the kidneys. Due to its synthesis, however, polylysine-GdDTPA can also contain macromolecules which are so large that they cannot pass through the renal capillaries during glomerular filtration and therefore remain in the body.
Macromolecular contrast media based on carbohydrates, for example dextran, have also been described (European Patent Application, Publication No. 0,326,226). The disadvantage of these compounds resides in that they carry normally only 4.6% of the signal-intensifying paramagnetic cation.
An object, therefore, resides in making available novel diagnostic aids, above all for the recognition and localization of vascular diseases, which aids do not exhibit the aforedescribed disadvantages. This and other objects have been attained by the present invention.
It has been found that complexes comprising nitrogen-containing cascade polymers provided with complexing ligands, ions of an element of atomic numbers 21-29, 39, 42, 44 or 57-83, as well as optionally cations of inorganic and/or organic bases, amino acids or amino acid amides, are surprisingly excellently suitable for the production of NMR and X-ray diagnostic media without exhibiting the afore-mentioned drawbacks.
The polymers according to this invention can be described by general Formula I 
wherein
A means a nitrogen-containing cascade nucleus of basis multiplicity b,
S means a reproduction unit,
N means a nitrogen atom,
Z1 and Z2, for the first to penultimate generation, in each case are 
xe2x80x83but, for the last generation,
Z means a hydrogen atom, a C1-C10-alkyl, C2-C10-acyl (e.g., alkanoyl) or C1-C10-alkylsulfonyl residue, each optionally containing 1-3 carboxy, 1-3 sulfonic acid, 1-5 hydroxy groups and/or 1-3 oxygen atoms (e.g., oxa (xe2x80x94Oxe2x80x94) atoms), or it means the residue of a complexing agent or complex K, and
means, to an extent of 96-100%, the residue of a complexing agent or complex K and, to an extent of 4-0%, Vxe2x80x2 wherein Vxe2x80x2 is the residue V exhibiting at the end a functional group or, linked via this functional group, a bio- or macromolecule, V meaning a straight-chain, branched, saturated or unsaturated C1-C20-alkylene group which optionally contains imino, phenylene, phenylenoxy, phenylenimino, amide, hydrazide, ureido, theoureido, carbonyl, ester group(s), oxygen, sulfur and/or nitrogen atom(s) and is optionally substituted by hydroxy, mercapto, imino, epoxy, oxo, thioxo, and/or amino group(s),
b means the numbers 1 through 50, and
s means the numbers 1 to 3,
wherein the reproduction units S can be different from generation to generation. Also the complex (forming) residues optionally standing for Z1 and Z2 need not be identical. A xe2x80x9cgenerationxe2x80x9d is represented by each S group in a chain of S groups.
Examples of alkyl, acyl and alkylsulfonyl residues standing for Z1 that can be cited are:
xe2x80x94CH2COOH; xe2x80x94(CH2)2COOH; xe2x80x94CH(COOH)CH2COOH; xe2x80x94CH2xe2x80x94CH(COOH)CH2OH; xe2x80x94CH2SO3H; xe2x80x94(CH2)2SO3H; xe2x80x94COCH3; xe2x80x94COCH2OH; xe2x80x94COCHOHCH2OH; xe2x80x94COCH2Oxe2x80x94CH2COOH; xe2x80x94CO(CHOH)4CH2OH; xe2x80x94COCH2COOH; xe2x80x94CO(CH2)COOH; xe2x80x94CO(CH2)3COOH; xe2x80x94CO(CH2)COOH; xe2x80x94COCHOHCOOH; xe2x80x94CO(CHOH)2COOH; xe2x80x94COCH2CHOHCH2COOH; xe2x80x94SO2CH2COOH; xe2x80x94SO2(CH2)COOH; xe2x80x94SO2CH3.
Suitable as the cascade nucleus A are:
a nitrogen atom, 
wherein
R2, R3 and R4 mean, in each case independently of one another, a covalent bond or 
g means the number 2, 3, 4 or 5,
t means the number 1, 2, 3, 4, 5, 6, 7 or 8,
l means the number 0, 1, 2, 3, 4 or 5,
r means the number 0 or 1,
n means the number 0, 1, 2, 3 or 4,
m means the number 0, 1, 2, 3 or 4,
k means the number 1, 2, 3, 4 or 5,
a means the number 2, 3, 4 or 5,
W means CH, CH2, NH or a nitrogen atom,
C1 means 
C2, C3, C4 and C5 mean, in each case independently, a hydrogen atom or 
f means the number 1, 2, 3, 4 or 5,
j means the number 6, 7 or 8,
Y1 and Y2 mean, in each case independently of each other, a hydrogen atom, 
xe2x80x83and
Y3 is a nitrogen atom, 
xe2x80x83a and g are as defined above,
 means a single or double bond,
with the proviso that, if Y3 is a nitrogen atom, Y1 and Y2 mean hydrogen. C6H4 is phenylene.
The simplest case of a cascade nucleus is represented by the nitrogen atom, the three bonds of which (basis multiplicity b=3) are occupied in a first xe2x80x9cinner layerxe2x80x9d (generation 1) by three reproduction units S each of which carries 1 to 3 terminal NH2 groups (s=1-3) (or, alternatively, the three hydrogen atoms of the basic cascade starter ammonia have been substituted by three units S). If the reproduction unit S contains, for example, an NH2 group (s=1), then the reproduction multiplicity of this generation is 2 s=2. The second layer (generation 2) of reproduction units S introduced in a subsequent reaction sequence (occupying, in the above-mentioned example with A=nitrogen atom and s=1, six bonds) need not be identical with the reproduction units S of the first generation. After preferably maximally 10, most preferably 2-6 generations, the terminal nitrogen atoms of the outermost layer are substituted as indicated above for Z1 and Z2 of the final generation.
Further preferred cascade starters A(H)b that can be listed are, inter alia:
tris(aminoethyl)amine (b=6);
tris(aminopropyl)amine (b=6);
diethylenetriamine (b=5);
triethylenetetramine (b=6);
tetraethylenepentamine (b=7),
H2Nxe2x80x94CH2xe2x80x94C6H4xe2x80x94CH2xe2x80x94NHxe2x80x94CH2xe2x80x94C6H4xe2x80x94NH2 (b=5):
1,3,5-tris(aminomethyl)benzene (b=6);
2,4,6-tris(aminomethyl)pyridine (b=6);
1,4,7-triazacyclononane (b=3);
1,4,7,10-tetraazacyclododecane (b=4);
1,4,7,10,13-pentaazacyclopentadecane (b=5);
1,4,8,11-tetraazacyclotetradecane (b=4);
1,4,7,10,13,16,19,22,25,28-decaazacyclotriacontane (b=10);
6,6xe2x80x2,6xe2x80x3,6xe2x80x2xe2x80x3,6xe2x80x3xe2x80x3,6xe2x80x2xe2x80x3xe2x80x3-hexaamino-6,6xe2x80x2,6xe2x80x3,6xe2x80x2xe2x80x3-6xe2x80x3xe2x80x3,6xe2x80x2xe2x80x3xe2x80x3-hexadeoxy-xcex1-cyclodextrin (b=12);
6,6xe2x80x2,6xe2x80x3,6xe2x80x2xe2x80x3,6xe2x80x3xe2x80x3,6xe2x80x2xe2x80x3xe2x80x3,6xe2x80x3xe2x80x3xe2x80x3-heptaamino-6,6xe2x80x2,6xe2x80x3,6xe2x80x2xe2x80x3,6xe2x80x3xe2x80x3,6xe2x80x2xe2x80x3xe2x80x3,6xe2x80x3xe2x80x3xe2x80x3-heptadeoxy-xcex2-cyclodextrin (b=14);
6,6xe2x80x2,6xe2x80x3,6xe2x80x2xe2x80x3,6xe2x80x3xe2x80x3,6xe2x80x2xe2x80x3xe2x80x3-hexa-(1-amino-2-hydroxy-propyl)-xcex1-cyclodextrin hexaether (b=12);
2,2xe2x80x2,2xe2x80x3,2xe2x80x2xe2x80x3,2xe2x80x3xe2x80x3,2xe2x80x2xe2x80x3xe2x80x3,6,6xe2x80x2,6xe2x80x3,6xe2x80x2xe2x80x3,6xe2x80x3xe2x80x3,6xe2x80x2xe2x80x3xe2x80x3-dodeca-(1-amino-2-hydroxypropyl)-xcex1-cyclodextrin dodecaether (b=24).
Thus, b generally is the number of cascadable (reacting) nitrogen valences (bonds) in an A group, e.g., corresponding to the number of H atoms bonded to N atoms.
The reproduction unit S typically has the formula
xe2x80x94(CH2)2xe2x80x94CONHxe2x80x94(CH2)a or 
wherein
a is a number 2, 3, 4 or 5,
xcex1 and xcex2 in each case mean a hydrogen atom or (CH2)o,
xcex3 means (CH2)f,
k is 1, 2, 3, 4 or 5,
l is 0, 1, 2, 3, 4 or 5,
o is 0, 1, 2, 3, 4 or 5,
f means the number 1, 2, 3, 4 or 5, and
r means the number 0 or 1,
with the proviso that o and 1 are not both zero at the same time.
Preferred reproduction units S are
xe2x80x94(CH2)2xe2x80x94CONHxe2x80x94(CH2)2xe2x80x94;
xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94;
xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94; 
Suitable complex (forming) residues K are described by general Formulae I A, I B and I C: 
wherein
n and m in each case independently mean the number 0, 1, 2, 3 or 4, n and m adding up to no more than 4,
k means the number 1, 2, 3, 4 or 5,
l means the number 0, 1, 2, 3, 4 or 5,
q means the number 0, 1 or 2,
u is CH2X or V,
X means in each case independently the residue xe2x80x94COOH or Vxe2x80x2 wherein, if the molecule contains Vxe2x80x2, at least 0.1% of the substituents X stand for Vxe2x80x2,
B, D and E, being identical or different, mean in each case the group xe2x80x94(CH2)a with a meaning the number 2, 3, 4 or 5,
R1 means V or hydrogen atom,
V and Vxe2x80x2 are as defined above,
with the proviso that R1 means V only if U means CH2X at the same time, and that U means V only if R1 means a hydrogen atom at the same time, as well as with the proviso that, if desired, a portion of the COOH groups is present as ester and/or amide.
Examples that can be cited for the complex forming residues K are those of ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, trans-1,2-cyclo-hexanediaminetetra-acetic acid, 1,4,7,10-tetraaza-cyclododecanetetraacetic acid, 1,4,7-triazacyclo-nonanetriacetic acid, 1,4,8,11-tetraazatetradecane-tetraacetic acid, 1,5,9-triazacyclododecanetriacetic acid, 1,4,7,10-tetraazacyclododecanetriacetic acid and 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-trienetriacetic acid which are linked via (in each case contained in K) a carbonyl group (I A; I B and I C, contained in V, e.g., where U is V if R1 means a hydrogen atom at the same time) or via a carbon atom (contained in V, see definition for U and R1 as per above, e.g., in I B and I C where R1 is V if U stands for CH2X at the same time) to respectively one terminal xe2x80x94NH2 group of the final generation of the cascade polymer. If desired, a portion of the carboxylic acids can be present as converted to ester and/or amide groups.
As Z2 of the last generation, Vxe2x80x2 can also be present up to a proportion of 4%.
Suitable complexes for Z1 and Z2 correspond to the foregoing complexing (chelating) agents as bonded (chelated) to central metal ions.
If the medium of this invention is intended for use in NMR diagnostics, then the central ion of the complex salt must be paramagnetic. These are, in particular, the di- and trivalent ions of the elements of atomic numbers 21-29, 42, 44 and 58-70. Suitable ions are, for example, the chromium(III), manganese(II), iron(II), cobalt(II), nickel(II), copper(II), praseodymium(III), neodymium(III), samarium(III) and ytterbium(III) ions. On account of their very strong magnetic moment, gadolinium(III), terbium(III), dysprosium(III), holmium(III), erbium(III) and iron(III) ions are especially preferred.
In case the agent of this invention is meant for use in X-ray diagnostics, the central ion must be derived from an element of a higher atomic number in order to obtain adequate absorption of the X rays. It has been found that diagnostic aids are suitable for this purpose which contain a physiologically compatible complex salt with central ions of elements of atomic numbers between 21-29, 39, 42, 44, 57-83; these are, for example, the lanthanum(III) ion and the above-mentioned ions of the lanthanide series.
The cascade polymer complexes according to this invention contain at least five of the ions of an element of the aforementioned atomic numbers.
The alkylene group standing for V as well as the hydrocarbyl group standing for R and Rxe2x80x2 (below) can be straight-chain, branched, cyclic, aliphatic, aromatic or arylaliphatic and can contain up to 20 carbon atoms. Straight-chain mono- to decamethylene groups as well as C1-C4-alkylenephenyl groups are preferred. The following alkylene groups are cited as examples for explanatory purposes: 
wherein R+ and Ry stand for natural amino acid residues;
xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)xe2x80x94NHCSxe2x80x94;
xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94NHCSxe2x80x94;
xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94Oxe2x80x94(CH2)2NHCSxe2x80x94;
xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NHxe2x80x94COxe2x80x94CH2xe2x80x94;
xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94Oxe2x80x94C6H4xe2x80x94NHCSxe2x80x94;
xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94Oxe2x80x94C6H4xe2x80x94NHCOxe2x80x94;
xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94C6H4xe2x80x94NHCSxe2x80x94;
xe2x80x94CH2xe2x80x94Oxe2x80x94C6H4xe2x80x94CH2xe2x80x94; xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94Oxe2x80x94C6H4xe2x80x94CH2;
xe2x80x94C(xe2x95x90NH)xe2x80x94Oxe2x80x94C6H4xe2x80x94CH2xe2x80x94;
xe2x80x94(CH2)4xe2x80x94NHxe2x80x94COxe2x80x94CH2xe2x80x94Oxe2x80x94C6H4xe2x80x94CH2xe2x80x94; xe2x80x94(CH2)4xe2x80x94NHxe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94Oxe2x80x94C6H4xe2x80x94CH2xe2x80x94;
xe2x80x94(CH2)3xe2x80x94Oxe2x80x94C6H4xe2x80x94CH2xe2x80x94; xe2x80x94CH2xe2x80x94COxe2x80x94NHxe2x80x94(CH2)3xe2x80x94Oxe2x80x94CH2xe2x80x94; xe2x80x94CH2xe2x80x94COxe2x80x94NHxe2x80x94NHxe2x80x94;
xe2x80x94CH2xe2x80x94CONHxe2x80x94(CH2)2xe2x80x94; xe2x80x94CH2xe2x80x94COxe2x80x94NHxe2x80x94(CH2)10xe2x80x94; xe2x80x94CH2xe2x80x94CONHxe2x80x94(CH2)2xe2x80x94Sxe2x80x94;
xe2x80x94(CH2)4xe2x80x94NHxe2x80x94COxe2x80x94(CH2)8xe2x80x94; xe2x80x94CH2xe2x80x94COxe2x80x94NHxe2x80x94(CH2)3xe2x80x94NHxe2x80x94; xe2x80x94(CH2)3xe2x80x94NHxe2x80x94 groups.
Preferred functional groups located at the end of the Vxe2x80x2 alkylene group are, for example, the maleimidobenzoyl, 3-sulfomaleimidobenzoyl, 4-(maleimidomethyl)cyclohexylcarbonyl, 4-[3-sulfo-(maleimidomethyl)cyclohexyl]carbonyl, 4-(p-maleimido-phenyl)butyryl, 3-(2-pyridyldithio)propionyl, methacryloyl (pentamethylene) amido, bromoacetyl, iodoacetyl, 3-iodopropyl, 2-bromoethyl, 3-mercapto-propyl, 2-mercaptoethyl, phenyleneisothiocyanate, 3-aminopropyl, benzyl ester, ethyl ester, tert-butyl ester, amino, C1-C6-alkylamino, aminocarbonyl, hydrazino, hydrazinocarbonyl, maleimido, methacryl-amido, methacryloylhydrazinocarbonyl maleimidamidocarbonyl, halogeno, mercapto, hydrazinotrimethylene-hydrazinocarbonyl, aminodimethyleneamidocarbonyl, bromocarbonyl, phenylenediazonium, isothiocyanate, semicarbazide, thiosemicarbazide, isocyanate groups.
Several selected groups will be set forth for explanatory purposes: 
wherein R and Rxe2x80x2 are identical or different and mean in each case a hydrogen atom, a saturated or unsaturated C1-C20-hydrocarbyl residue optionally sub-stituted by a phenyl group, or a phenyl group.
The residual acidic hydrogen atoms, i.e. those that have not been substituted by the central ion, can be replaced, if desired, entirely or partially by cations of inorganic and/or organic bases or amino acids. The corresponding acid groups can also be converted entirely or partially into esters or amides.
Suitable inorganic cations are, for example, the lithium ion, the potassium ion, the calcium ion, the magnesium ion, and especially the sodium ion. Suitable cations of organic bases are, inter alia, those of primary, secondary or tertiary amines, e.g. ethanolamine, diethanolamine, morpholine, glucamine, N,N-dimethylglucamine and, in particular, N-methylglucamine. Suitable cations of amino acids are, for example, those of lysine, of arginine and of ornithine, as well as the amides of otherwise acidic or neutral amino acids.
Suitable esters are preferably those with a C1-C6-alkyl residue; examples that can be cited are the methyl, ethyl and tert-butyl residues.
In case the carboxylic acid groups are to be present at least in part as amides, then tertiary amides are preferred. Suitable residues are saturated, unsaturated, straight- or branched-chain or cyclic hydrocarbons of up to 5 carbon atoms optionally substituted by 1-3 hydroxy or C1-C4-alkoxy groups. Examples that can be cited are the methyl, ethyl, 2-hydroxyethyl, 2-hydroxy-1-(hydroxymethyl)ethyl, 1-(hydroxymethyl)ethyl, propyl, isopropenyl, 2-hydroxypropyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, butyl, isobutyl, isobutenyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2-, 3- and 4-hydroxy-2-methylbutyl, 2- and 3-hydroxyisobutyl, 2,3,4-trihydroxybutyl, 1,2,4-trihydroxybutyl, pentyl, cyclopentyl and 2-methoxyethyl groups. The amide residue can also be a heterocyclic 5- or 6-membered ring formed with inclusion of the amide nitrogen. Examples in this connection are: the pyrrolidinyl, piperidyl, pyrazolidinyl, pyrrolinyl, pyrazolinyl, piperazinyl, morpholinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl rings.
The compounds of this invention exhibit the desirable properties set forth hereinabove. They contain the large number of metal ions, required for their use, bound in the complex in a stable fashion. They are distributed (if Vxe2x80x2 does not contain a bio- or macromolecule) only in the vasal space and thus can map this space with the aid of nuclear spin tomography.
The compatibility of the compounds according to this invention is improved by at least a factor of 3 over xe2x80x9cMagnevistxe2x80x9d (LD50 i.v. mice of Example 8: 30 mmol/kg; xe2x80x9cMagnevistxe2x80x9d: xe2x89xa610).
The value of osmolality, responsible for side effects, such as pain, damage to the blood vessels and cardiovascular disturbances, is. reduced as compared with xe2x80x9cMagnevistxe2x80x9d (Example 8: 0.46 [osmol/kg] as compared with xe2x80x9cMagnevistxe2x80x9d 1.96 [osmol/kg], 0.5 mol/l at 37xc2x0 C.).
The value for the magnitude of relaxation, representing a measure for imaging in MRI, is surprisingly high; signal intensification could be increased over xe2x80x9cMagnevistxe2x80x9d, for example in case of the compound of Example 8, fourfold.
As compared with the macromolecular contrast media based on carbohydrates, e.g. dextran (European Patent Application, Publication No. 0,326,226) which carryxe2x80x94as mentionedxe2x80x94normally only 4.6% of the signal-intensifying paramagnetic cation, the polymer complexes of this invention contain more than 15% of the paramagnetic cation. Accordingly, the macromolecules of this invention bring about, per molecule, a very much higher signal intensification which has the result, at the same time, that the dose required for nuclear spin tomography is considerably smaller as compared with that for macromolecular contrast media based on carbohydrates.
It has been made possible with the polymer complexes according to this invention to construct and produce macromolecules in such a way that they exhibit a uniformly defined molecular weight. Such macromolecular contrast media, exactly definable in their molecular size, have not been accessible heretofore. It is thus surprisingly possible for the first time to regulate the size of the macromolecules so that these are large enough to be able to leave the vasal space only gradually but, at the same time, small enough to still pass through the kidney capillaries which have a size of 300-800 xc3x85. It has thus been accomplished for the first time to produce macromolecular contrast media tailored to the body.
The complexes of this invention serve as contrast media for imaging the vessels by means of nuclear spin tomography. It is thus possible to differentiate between ischemic tissue and normal tissue. However, also other damage to the blood-tissue barrier can be recognized by means of these compounds. In case of inflammations and tumors in the brain, the blood-brain barrier is damaged to such an extent that the contrast medium can infiltrate the diseased tissue and thus the diseased tissue becomes visible in nuclear spin tomography. On account of the impermeability of the intact blood-brain barrier, even to small, but hydrophilic molecules, inflammations and tumors have also been recognizable even with the low-molecular compound xe2x80x9cMagnevistxe2x80x9d. However, when using the complexes of this invention in these cases, the dosage can be reduced sixteenfold, for two reasons: (1) they exhibit a signal intensification which is four times higher, and (2) they are distributed in a space that is four times smaller, namely only in the vasal space, i.e. in order to reach the same concentrations in the blood, one-fourth of the dose is sufficient.
Another advantage of the present invention resides in that complexes with hydrophilic or lipophilic, macrocyclic or open-chain, low-molecular or high-molecular ligands have now become accessible. This affords the possibility of controlling compatibility and pharmacokinetics of these polymer complexes by chemical substitution.
By the choice of suitable bio- or macromolecules (see further below) in Vxe2x80x2, polymer complexes according to this invention are obtained which exhibit a surprisingly high tissue and organ specificity.
The cascade polymers according to this invention are produced by reacting compounds of general Formula Ixe2x80x2
wherein
A means a nitrogen-containing cascade nucleus of the basis multiplicity b,
S means a reproduction unit,
N means a nitrogen atom,
Z1xe2x80x2 and Z2xe2x80x2 mean the first to penultimate generation, in each case 
xe2x80x83but, for the final generation, in each case mean a hydrogen atom,
b means the numbers 1 through 50, and
s means the numbers 1 to 3,
wherein the reproduction units S need to be identical only for one generationxe2x80x94optionally after reaction of up to 4% of the terminal amino groups with a C4-C20-alkylene chain that is substituted at the ends by carboxyl and hydrazide (preferably in the blocked form)xe2x80x94
with a complex or complexing compound Kxe2x80x2 of the general formulae 
wherein
n and m in each case are the number 0, 1, 2, 3 or 4, n and m adding up to no more than 4,
k means the number 1, 2, 3, 4 or 5,
l means the number 0, 1, 2, 3, 4 or 5,
q means the number 0, 1 or 2,
Uxe2x80x2 means xe2x80x94CH2C*Oxe2x80x94, CH2Xxe2x80x2 or Vxe2x80x3 wherein Vxe2x80x3 stands for a straight-chain, branched, saturated or unsaturated C1-C20-alkylene group which optionally contains imino, phenylene, phenylenoxy, phenylenimino, amide, hydrazide, ureido, thioureido, carbonyl, ester group(s), oxygen, sulfur and/or nitrogen atom(s) and is optionally substituted by hydroxy, mercapto, imino, epoxy, oxo, thioxo and/or amino group(s), this alkylene group carrying a functional group at the end,
Xxe2x80x2 means in each case independently the residues xe2x80x94COOH, COOY or Vxe2x80x2xe2x80x3,
wherein
Y is an acid blocking group or a metal ion equivalent of an element of atomic numbers 21-29, 39, 42, 44 or 57-83, and
Vxe2x80x2xe2x80x3 means a substituent to be converted into Vxe2x80x2,
C*O stands for an activated carbonyl group,
B, D and E, being identical or different, mean in each case the group (CH2)a where a means the number 2, 3, 4 or 5,
R1xe2x80x2 is Vxe2x80x3 or a hydrogen atom,
with the proviso that R1xe2x80x2 stands for Vxe2x80x3 only if Uxe2x80x2 means CH2Xxe2x80x2 at the same time, and that Uxe2x80x2 means xe2x80x94CH2C*Oxe2x80x94 or Vxe2x80x3 only if R1xe2x80x2 means a hydrogen atom at the same time,
optionally splitting off any blocking groups present, reacting, if desired, the thus-obtained cascade polymersxe2x80x94insofar as Kxe2x80x2 means a complexing compoundxe2x80x94conventionally with at least one metal oxide or metal salt of an element of atomic numbers 21-29, 39, 42, 44 or 57-83, and optionally converting them into the cascade polymers carrying the desired macro- or biomolecule(s) by conversion of at least one of the xe2x80x94CO2Hxe2x80x94 or Vxe2x80x2xe2x80x3 groups contained in Kxe2x80x2 into the desired alkylene group Vxe2x80x3 exhibiting a functional group at the end and optionally by subsequent linkage via this functional group and/or via the terminal-positioned hydrazide group that may be contained in Z2, with a macro- or biomolecule and/or by linkage to the biotin or avidin residue, wherein the indicated reaction steps (except for the macro- or biomolecule linkage which can take place only after generating the functional group) can be performed in any desired sequence, and optionally substituting, subsequently, in the thus obtained polymer complexes any still present acidic hydrogen atoms entirely or partially by cations of inorganic and/or organic bases, amino acids or amino acid amides or converting the corresponding acid groups entirely or partially into esters or amides.
Examples of an activated carbonyl group in the complexes and/or complex-forming compounds Kxe2x80x2 are anhydride, p-nitrophenyl ester and acid chloride.
The alkylation or acylation effected for the introduction of the complex-forming units is carried out with substrates containing the desired substituent K (possibly bound to a leaving group), or from which the desired substituent, optionally after modification by secondary reaction(s), is generated by the reaction. Examples that can be cited for the first-mentioned substrates are halogenides, mesylates, tosylates and anhydrides. Among the second group are, for example, oxiranes, thiiranes, aziranes, xcex1,xcex2-unsaturated carbonyl compounds or their vinylogs, aldehydes, ketones, isothiocyanates and isocyanates.
Examples of secondary reactions that can be mentioned are ester cleavages, hydrogenations, esterifications, oxidations, etherifications and alkylations, performed in accordance with literature methods known to those skilled in the art.
Selected examples of the residues Vxe2x80x3 contained in Kxe2x80x2 are listed as follows:
xe2x80x94CH2xe2x80x94CHOHxe2x80x94CH2xe2x80x94NCS;
xe2x80x94CH2xe2x80x94CHOHxe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NCS;
xe2x80x94CH2xe2x80x94CHOHxe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NHxe2x80x94COxe2x80x94CH2xe2x80x94Br;
xe2x80x94CH2xe2x80x94CHOHxe2x80x94CH2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94Oxe2x80x94(CH2)2xe2x80x94NCS;
xe2x80x94NCS;
xe2x80x94CH2xe2x80x94CHOHxe2x80x94CH2xe2x80x94Oxe2x80x94CH xe2x80x94CHO;
xe2x80x94CH2xe2x80x94CHOHxe2x80x94CHO; 
An example that can be cited is the reaction of the monoanhydride N3-(2,6-dioxomorpholinoethyl)-N6-(ethoxycarbonylmnethyl)-3,6-diazaoctanedioic acid with the respectively desired cascade polymers, containing terminal amino groups, in water or in mixtures of water with, for example, dioxane, THF, DMF, DMSO or acetonitrile at an alkaline pH, preferably 8-10, i.e. with the addition of bases, such as, for example, sodium hydroxide, potassium hydroxide or triethylamine, at temperatures of 0-50xc2x0 C., preferably at room temperature. For a complete reaction, a two- to threefold excess of monoanhydride, for example, is preferably employed.
A further possibility that can be mentioned is the reaction of substituents Kxe2x80x2 exhibiting terminal-positioned aldehyde groups with the respectively desired cascade polymers containing terminal amino groups, with subsequent reduction of the thus-formed Schiff bases analogously to methods known from the literature (Synthesis 1975, 135). The thus-generated secondary amines can be converted into tertiary; amines, amides or thioamides by subsequent acylation or alkylation with xcex1,xcex2-unsaturated esters containing optionally 1-3 carboxy, 1-3 sulfonic acid, 1-5 hydroxy residues and/or 1-3 oxygen atoms, alkyl halogenides, anhydrides, acid halogenides, or complexes and/or complexing compounds Kxe2x80x2. As examples of reaction partners which substitute the secondary amino hydrogen atoms the following can be cited:
Brxe2x80x94CH2COOH; Clxe2x80x94CH2xe2x80x94CH xe2x80x94COOH; H2Cxe2x95x90CHxe2x80x94COOCH3; HOOCxe2x80x94CHxe2x95x90CHxe2x80x94COCCH3; CH2xe2x95x90C(COO"Egr"t)xe2x80x94CH2OH; Brxe2x80x94CH2xe2x80x94So3H; Clxe2x80x94CH2xe2x80x94CH2xe2x80x94SO3H; CH3xe2x80x94COxe2x80x94Cl; Clxe2x80x94COxe2x80x94CH2OH; Clxe2x80x94COxe2x80x94CHOHxe2x80x94CH2OH; ClSO2CH3; ClSO2CH2COOC2H5; Brxe2x80x94COxe2x80x94(CHOH)4xe2x80x94C2OH; Clxe2x80x94COxe2x80x94CH2xe2x80x94COOC2H5; 
xe2x80x83Brxe2x80x94COxe2x80x94(CH2)4xe2x80x94COOC(CH3)3; Clxe2x80x94COxe2x80x94CHOHxe2x80x94COOCH3.
The aldehydes required herein as educts can be prepared from the corresponding vicinal diols by oxidation with, for example, sodium metaperiodate in an aqueous or alcoholic solution analogously to methods known from the literature (e.g. xe2x80x9cMakromol. Chem.xe2x80x9d 182:1641 [1981]).
By pursuing a suitable course of reaction, for example adjusting the pH value or addition of amines, concomitantly introduced ester groups can, if desired, be saponified and aminolyzed, respectively.
Purification of the resultant cascade polymers takes place preferably by ultrafiltration with membranes of a suitable pore size (e.g. xe2x80x9cAmiconxe2x80x9d) or gel filtration on, for example, suitable xe2x80x9cSephadexxe2x80x9d gels.
Analogously, for example, complexing compounds or complexes, derived from isothiocyanate, epoxide or a-halogenoacetyl, are made to react under pH control in an aqueous medium with the desired cascade polymer amines
The compounds Ixe2x80x2 required as the educts are known (for example, European Patent Applications, Publication Nos. 0,154,788 and 0,331,616, German Patent Application P 38 25 040.3) or they can be prepared from the corresponding polyamines (wherein any present functional groups are optionally blocked) by alkylation with an ester of general Formula II
HalCH2COOYxe2x80x2xe2x80x83xe2x80x83(II)
wherein Hal means chlorine, bromine or iodine and Yxe2x80x2 means a hydrogen atom, an alkali metal or an acid blocking group Y.
The reaction takes place in polar aprotic solvents, such as, for example, dimethylformamide, dimethyl sulfoxide, acetonitrile, aqueous tetrahydro-furan or hexamethylphosphoric triamide in the presence of an acid captor, such as, for example, a tertiary amine (e.g. triethylamine, trimethylamine, N,N-dimethylaminopyridine, 1,5-diazabicycl[4.3.0]nonene-5 (DBN), 1,5-diazabicycl[5.4.0]undecene-5 (DBU), alkali, alkaline earth carbonate, bicarbonate or hydroxide (e.g. sodium, magnesium, calcium, barium, potassium carbonate, hydroxide and bicarbonate) at temperatures of between xe2x88x9210xc2x0 C. and 120xc2x0 C., preferably between 0xc2x0 C. and 50xc2x0 C.
Suitable acid blocking groups Y are lower alkyl, aryl and aralkyl groups, for example the methyl, ethyl, propyl, butyl, phenyl, benzyl, diphenylmethyl, triphenylmethyl, bis(p-nitrophenyl)-methyl groups, as well as trialkylsilyl groups.
The splitting off of the blocking groups Y which may be desirable takes place according to the methods known to one skilled in the art, for example by hydrolysis, hydrogenolysis, alkaline saponification of the esters with an alkali in an aqueous-alcoholic solution at temperatures of 0xc2x0 C. to 50xc2x0 C. or, in case of tert-butyl esters, with the aid of trifluoroacetic acid.
Production of the derivatives with an activated carbonyl group C*O, Ixe2x80x2A or Ixe2x80x2B and Ixe2x80x2C wherein Uxe2x80x2 means CH2C*O (e.g. mixed anhydride, N-hydroxy-succinimide ester, acylimidazoles, trimethylsilyl ester) takes place according to methods known from the literature [Houben-Weyl, xe2x80x9cMethoden der organischen Chemiexe2x80x9d [Methods of Organic Chemistry], Georg Thieme publishers, Stuttgart, vol. E 5 (1985), 633; Org. React. 12:157 (1962)] or will be described in the experimental portion.
Preparation of the cyclic polyamines needed as educts for Ixe2x80x2B and Ixe2x80x2C takes place by cyclization of two reactants of whichxe2x80x94in case of the synthesis of Ixe2x80x2B with R1xe2x80x2=Vxe2x80x3xe2x80x94one is Vxe2x80x2xe2x80x3-substituted, or (in case of the synthesis of Ixe2x80x2C) one contains the desired 6-membered ring of the final product or a precursor to be converted into this ring.
The cyclization is carried out according to methods known from the literature [for example, Org. Synth. 51:86 (1978), Macrocyclic Polyether Syntheses, Springer publishers, Berlin, Heidelberg, N.Y. (1982), Coord. Chem. Rev. 3:3 (1968), Ann. Chem. 1976 916, J. Org. Chem. 49 110 (1984)]; one of the two reactants carries two leaving groups at the chain end, the other carries two nitrogen atoms which displace these leaving groups in nucleophilic fashion. An example that can be cited is the reaction of terminal-positioned dichloro, dibromo, dimesyloxy, ditosyloxy or dialkoxycarbonyl alkylene compounds, containing, if desired, the substituent Vxe2x80x2xe2x80x3 and optionally one to five nitrogen atom(s), with terminal-positioned polyazaalkylene compounds optionally containing one to five additional nitrogen atom(s) in the alkylene chain. The substituent Vxe2x80x2xe2x80x3 can, instead, also be contained in the second reactant, i.e. the one having the terminal-positioned nucleophilic nitrogen atoms. The nitrogen atoms are blocked, if necessary, for example as tosylates or trifluoroacetates, and they are liberated according to methods known in the literature prior to the subsequent alkylation reaction (the tosylates, for example, with mineral acids, alkali metals in liquid ammonia, hydrobromic acid and phenol, xe2x80x9cRedAlxe2x80x9d, lithium aluminum hydride, sodium amalgam, compare, for example, Liebigs Ann. Chem. 1977:1344, Tetrahedron Letters 1976:3477; the trifluoroacetates, for example, with mineral acids or ammonia in methanol, compare, for example, Tetrahedron Letters 1967:289).
For preparing macrocycles differently substituted on the nitrogen atoms (hydrogen or the group CH2COOY), these atoms can be provided in the educts with differing blocking groups, for example with tosylate and benzyl groups. The latter are then likewise removed according to methods known in the literature (preferably by hydrogenation, e.g. EP Patent Application 232,751).
In case diesters are used in the cyclization reaction, the resultant diketo compounds must be reduced by methods known to a person skilled in the art, for example with diborane.
It is also possible to cyclize correspondingly substituted terminal-positioned bisaldehydes with the respectively desired terminal-positioned bisamines; the reduction of the thus-obtained Schiff bases takes place according to methods known in the literature, for example by catalytic hydrogenation [Helv. Chim. Acta 61 :1376 (1978)].
The amines required to serve as starting materials for the cyclization are prepared in analogy to methods known from the literature.
Starting with an N-blocked amino acid, reaction with a partially blocked diamine (e.g. according to the carbodiimide method), splitting off of the blocking groups, and diborane reduction yield a triamine.
Reaction of a diamine obtained from amino acids [Eur. J. Med. Chem. -Chim. Ther. 21:333 (1986)] with twice the molar amount of an N-protected xcfx89-amino acid yields a tetramine after appropriate working up procedure.
The desired diamines can also be prepared by Gabriel reaction from, for example, the corresponding tosylates or halogenides [compare, for example, Inorg. Chem. 25:4781 (1986)].
In both cases, the number of carbon atoms between the N atoms can be determined by the type of diamines or amino acids utilized as coupling partners.
Conversion of a precursor of Ixe2x80x2C, obtained by cyclizing, into the desired complexing compound takes place according to methods known to one skilled in the art, for example deoxygenation of nitroxide [E. Klingsberg, The Chemistry of Heterocyclic Compounds, vol. 14, part 2, Interscience Publishers, New York, page 120, 1961) rings, conversions, and introduction of functional groups at the pyridine ring, e.g. liberation of phenolic hydroxy groups (J. Org. Chem. 53:5 (1988)], introduction of halogen substituents [E. Klingsberg, The Chemistry of Heterocyclic Compounds, vol. 14, part 2, Interscience Publishers, New York, page 341, 1961; Houben-Weyl, xe2x80x9cMethoden der organischen Chemiexe2x80x9d, vol. V/3, 651 (1962)].
Functionalization of 4-halopyridine derivatives (e.g. azide exchange) in the phase transfer process with the use of 18-crown-6 or tetrabutyl-ammonium halogenide as the catalyst has been described in xe2x80x9cPhase Transfer Reactionsxe2x80x9d (Fluka Compendium vol. 2, Walter E. Keller, Georg Thieme publishers, Stuttgart, N.Y.). A thus-obtained azide group can be converted into an amino function in accordance with methods known to one skilled in the art (e.g. catalytic hydrogenation, Houben-Weyl, xe2x80x9cMethoden der organischen Chemiexe2x80x9d, vol. 11/1, p. 539; or reaction with Raney nickel/hydrazine, German Patent Application 3,150,917). This amino function can be converted into an isothiocyanate group according to methods known from the literature (for example with thiophosgene in a two-phase system, S. Scharma, Synthesis 1978:803; D. K. Johnson, J. Med. Chem. 1989, vol. 32, 236).
By reacting an amino function with a halo-acetic acid halogenide, an xcex1-halogenoacetamide group can be generated (JACS 1969, vol. 90, 4508; Chem. Pharm. Bull. 29 (1) 128, 1981) which is suitable for coupling to bio- or macromolecules or cascade polymers in the same way as, for example, the isothiocyanate group.
As a substituent Vxe2x80x2xe2x80x3 which can be converted into V, or into the substituent Vxe2x80x3 exhibiting at the end a functional group suitable for linking to a macro- or biomolecule or to a cascade polymer, suitable are, inter alia, hydroxy and nitrobenzyl, hydroxy and carboxyalkyl, as well as thioalkyl residues of up to 20 carbon atoms. They are converted, according to literature methods known to one skilled in the art [Chem. Pharm. Bull. 33:674 (1985), Compendium of Org. Synthesis. vol. 1-5, Wiley and Sons, Inc., Houben-Weyl, xe2x80x9cMethoden der organischen Chemiexe2x80x9d, vol. VIII, Georg Thieme publishers, Stuttgart, J. Biochem. 92:1413 (1982)], into the desired substituents (e.g. with the amino, hydrazino, hydrazinocarbonyl, epoxide, anhydride, methacryloylhydrazinocarbonyl, maleimidamidocarbonyl, halo, halocarbonyl, mercapto, isothiocyanate group as the functional group) where, in case of the nitrobenzyl residue, first a catalytic hydrogenation to the aminobenzyl derivative must be performed (for example according to P. N. Rylander, Catalytic Hydrogenation Over Platinum Metals, Academic Press, 1967).
Examples of the conversion of hydroxy or amino groups bound to aromatic or aliphatic residues are the reactions carried out with a substrate of general Formula III
Nf-L-Fuxe2x80x83xe2x80x83(III)
wherein
Nf means a nucleofugal entity, such as, for example, Cl, Br, I, CH3C6H4SO3 or CF3SO3,
L is an aliphatic, aromatic, arylaliphatic, branched, straight-chain or cyclic hydrocarbon residue of up to 20 carbon atoms, and
Fu is the desired, terminal-positioned functional group, optionally in the blocked form (DOS 3,417,413),
performed in suitable solvents, such as tetrahdyrofuran, dimethoxyethane or dimethyl sulfoxide, two-phase aqueous systems, such as, for example, water/dichloromethane, in the presence of an acid captor, such as, for example, sodium hydroxide, sodium hydride or alkali or alkaline earth carbonates, such as, for example, sodium, magnesium, potassium, calcium carbonate or poly-(4-vinylpyridine) xe2x80x9cReillexxe2x80x9d, at temperatures of between 0xc2x0 C. and the boiling point of the respective solvent, but preferably between 20xc2x0 C. and 60xc2x0 C.
Examples of compounds according to general Formula III are:
Br(CH2)2NH2, Br(CH2)3OH, BrCH2COOCH3, BrCH2CO2tBu, ClCH2CONHNH2, Br(CH2)4CO2C2H5, BrCH2COBr, BrCH2CONH2, ClCH2COOC2H5, BrCH2CONHNH2, 
xe2x80x83CF3SO3(CH2)3Br, BrCH2Cxe2x89xa1CH, BrCH2CHxe2x95x90CH2.
Conversions of carboxy groups can be performed, for example, according to the carbodiimide method (Fieser, Reagents for Organic Syntheses 10, 142), by way of a mixed anhydride [Org. Prep. Proc. Int. 7:215 (1975)] or by way of an activated ester (Adv. Org. Chem., part B, 472).
Introduction of the optionally desired substituent Vxe2x80x3 at a nitrogen atom of the complexing compounds Ixe2x80x2B and Ixe2x80x2C (i.e., Uxe2x80x2=Vxe2x80x3) can likewise be effected according to the above-mentioned process, i.e. here, too, a macrocycle intermediate stage containing Vxe2x80x2xe2x80x3 is usually passed through which is obtained by reaction of a polyaza macrocycle exhibiting only one free NH group. Examples in this connection are the reaction of, for example, 1,4,7-triscarboxy-methyl-1,4,7,10-tetraazacyclocodecane with a primary epoxide exhibiting a blocked amino group, subsequent liberation of the amino function of the resultant Vxe2x80x2xe2x80x3-substituted macrocycle, and subsequent conversion into a Vxe2x80x3-substituted macrocycle (for example, conversion of the amino group into a functional group that can be coupled to the cascade polymer amine, such as, for example, the isothiocyanate or 2-halogeno-acetamide group).
The cascade polymers carrying terminal amino groups, needed for coupling to the complexing compounds K (and/or also the corresponding metal-containing complexes), are prepared according to methods known to persons skilled in the art by a cascade-type, generation-wise introduction of nitrogen atoms into a nitrogen-containing basis molecule. This yields a generation from at least two reaction steps. From each amino hydrogen atom of the cascade starter, up to three amino groups are generated in this way by, for example, a Michael addition or addition of a primary epoxide containing a suitable functional group, and subsequent conversion of the thus-introduced functional group.
An example that can be cited is the substitution of the six amino hydrogen atoms of the cascade starter tris(aminoethyl)amine by six xe2x80x94CH2CH2xe2x80x94CONHxe2x80x94CH2CH2NH2 units obtained by Michael addition with acrylic acid ester and subsequent aminolysis with ethylenediamine. The aminolysis, preferably performed without solvents, is here conducted with an up to 500-fold amine excess per ester grouping at temperatures of 0xc2x0 C. to about 130xc2x0 C.
As an example of an epoxide addition, the reaction can be cited of 6,6xe2x80x2,6xe2x80x3,6xe2x80x2xe2x80x3,6xe2x80x3xe2x80x3,6xe2x80x2xe2x80x3xe2x80x3-hexaamino-6,6xe2x80x2,6xe2x80x3,6xe2x80x2xe2x80x3,6xe2x80x3xe2x80x3,6xe2x80x2xe2x80x3xe2x80x3-hexadeoxy-xcex1-cyclodextrin with 1,3-(N,Nxe2x80x2-tetrabenzyl)diamino-2-(oxiranylmethoxy)propane and subsequent liberation of the amino functions by catalytic hydrogenation in accordance with methods known to one skilled in the art (see also above).
A portion of the acid groups of the thus-obtained polymer compounds, introduced via the complex forming units K, can be further functionalized, if desired, according to processes known to a person skilled in the art, for example by converting into ester, amide, hydrazide, maleimido or other groups suitable for coupling to bio- or macromolecules.
The thus-obtained complexing ligands (as well as the complexes) can also be linked to bio- or macromolecules from which it is known that they are particularly accumulated in the organ or organ part to be examined. Such molecules are, for example, enzymes, hormones, polysaccharides, such as dextrans or starches, porphyrins, bleomycins, insulin, prostaglandins, steroid hormones, amino sugars, amino acids, peptides such as polylysine, proteins (such as, for example, immunoglobulins, monoclonal antibodies, lectins), lipids (also in the form of liposomes), and nucleotides of the DNA or RNA type. Especially to be emphasized are conjugates with albumins, such as human serum albumin, antibodies, e.g. monoclonal antibodies specific for tumor-associated antigens, or antimyosin. Instead of biological macromolecules, it is also possible to link suitable synthetic polymers, such as polyethylenimines, polyamides, polyureas, polyethers, such as polyethylene glycols, and polythioureas. The pharmaceutical agents formed therefrom are suitabe, for example, for use in tumor and infarction diagnostics, as well as tumor therapy. Monoclonal antibodies (e.g. Nature 256:495, 1975) have the advantages over polyclonal antibodies that they are specific for an antigen determinant, that they possess definite binding affinity, that they are homogeneous (thus substantially simplifying their production in pure form), and that they can be manufactured in large amounts in cell cultures. Suitable are, for example, for tumor imaging, monoclonal antibodies and/or their fragments Fab and F(abxe2x80x2)2 which are specific, for example, for human tumors of the gastrointestinal tract, of the breast, of the liver, of the bladder, of the-gonads, and of melanomas [Cancer Treatment Repts. 68:317 (1984), Bio. Sci. 34:150 (1984)] or are directed against carcinomembryonal antigen (CEA), human chorionic gonadotropin (xcex2-HCG), or other tumor-positioned antigens, such as glycoproteins [New Engl. J. Med. 298:1384 (1973), U.S. Pat. No. 4,331,647]. Suitable are, inter alia, also antimyosin, anti-insulin and antifibrin antibodies (U.S. Pat. No. 4,036,945).
Colon carcinomas can be confirmed by NMR diagnosis with the aid of conjugates complexed with gadolinium(III) ions, using the antibody 17-1A (Centocor, USA).
For liver examinations and tumor diagnostics, respectively, conjugates or inclusion compounds are suitable, for example, with liposomes utilized, for instance, as unilamellar or multilamellar phosphatidylcholine cholesterol vesicles.
Heretofore, bonding of metals to the desired macro- or biomolecules has been performed according to methods described, for example, in Rev. Roum. Morphol. Embryol. Physio., Physiologie 1981, 18:241, and in J. Pharm. Sci. 68:79 (1979), e.g. by reaction of the nucleophilic group of a macromolecule, such as the amino, phenol, sulfhydryl, aldehyde or imidazole group, with an activated derivative of the polymer complex or ligand. Examples of activated derivatives are anhydrides, acid chlorides, mixed anhydrides (see, for example, G. E. Krejcarek and K. L. Tucker, Biochem., Biophys. Res. Commun. 1977, 581), activated esters, nitrenes or isothiocyanates. Conversely, it is also possible to react an activated macromolecule with the polymer complex or ligand. For conjugation with proteins, also suitable are, for example, substituents of the structure C6H2N2+, C6H4NHCOCH2Br, C6H4NCS or C6H4OCH2COBr.
However, this type of linkage is burdened by the drawback of lack in complex stability of the conjugates and/or lack of specificity (for instance, Diagnostic Imaging 84 56; Science 220:613, 1983; Cancer Drug Delivery 1:125, 1984). The conjugate formation according to the present invention takes place, in contrast thereto, via the functional groups present in Vxe2x80x2. It is possible herein to bind up to more than one-hundred metal ions via one binding site in the macromolecule.
In case of the antibody conjugates, binding of the antibody to the complex or ligand must not lead to loss or reduction of binding affinity and binding specificity of the antibody to the antigen. This can be accomplished either by binding to the carbohydrate portion in the Fc part of the glycoprotein and/or in the Fab or F(abxe2x80x2)2 fragments, or by binding to sulfur atoms of the antibody and/or antibody fragments.
In the first instance, an oxidative cleavage of sugar units must first be performed for the generation of formyl groups capable of coupling. This oxidation can be carried out by chemical methods with oxidizing agents such as, for example, periodic acid, sodium metaperiodate, or potassium metaperiodate in accordance with methods known from the literature (e.g., J. Histochem. and Cytochem. 22:1084, 1974) in an aqueous solution in concentrations of 1-100 mg/ml, preferably 1-20 mg/ml, and with a concentration of the oxidizing agent of between 0.001 to 10 millimoles, preferably 1 to 10 millimoles, in a pH range of about 4 to 8 at a temperature of between 0xc2x0 and 37xc2x0 C. and with a reaction period of between 15 minutes and 24 hours. The oxidation can also be performed by enzymatic methods, for example with the aid of galactose oxidase in an enzyme concentration of 10-100 units/ml, a substrate concentration of 1-20 mg/ml, at a pH of 5 to 8, a reaction period of 1-8 hours, and a temperature of between 20xc2x0 and 40xc2x0 C. (for example, J. Biol. Chem. 234:445, 1959).
Complexes or ligands with suitable functional groups, such as, for example hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide, are bound to the aldehydes generated by oxidation; this is done by reacting between 0xc2x0 and 37xc2x0 C. with a reaction period of 1-65 hours, a pH of between about 5.5 and 8, an antibody concentration of 0.5-20 mg/ml, and a molar ratio of the complexing compound to the antibody aldehyde of 1:1 to 1000:1. The subsequent stabilization of the conjugate takes place by reduction of the double bond, for example with sodium borohydride or sodium cyanoborohydride; the reducing agent is utilized herein with a 10- to 100-fold excess (e.g., J. Biol. Chem. 254:4359, 1979).
The second possibility of forming antibody conjugates starts with a gentle reduction of the disulfide bridges of the immunoglobulin molecule; in this process, the more sensitive disulfide bridges between the H chains of the antibody molecule are cleaved whereas the Sxe2x80x94S bonds of the antigen-binding region remain intact so that there is practically no reduction in binding affinity and specificity of the antibody (Biochem. 18:2226, 1979; Handbook of Experimental Immunology, vol. 1, 2nd ed., Blackwell Scientific Publications, London 1973, chapter 10). These free sulfhydryl groups of the inter-H-chain regions are then reacted with suitable functional groups of complexing compounds or metal complexes at 0-37xc2x0 C., a pH of about 4-7, and a reaction period of 3-72 hours with the formation of a covalent bond which does not affect the antigen binding region of the anti-body. Suitable reactive groups are, for example: haloalkyl, haloacetyl, p-mercuribenzoate, isothiocyanate, thiol, epoxy groups, as well as groups to be subjected to a Michael addition reaction, such as, for example, maleinimides, methacrylo groups (e.g. J. Amer. Chem. Soc. 101:3097, 1979).
Additionally, for linking the antibody fragments with the polymer complexes or with the ligands, there is a number of suitable bifunctional xe2x80x9clinkersxe2x80x9d which are frequently also obtainable commercially (see, for example, Pierce, Handbook and General Catalogue 1986) which are reactive with respect to the SH groups of the fragments as well as with respect to the amino or hydrazino groups of the polymers.
Examples that can be cited are:
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
m-maleimidobenzoyl-N-sulfosuccinimide ester (Sulfo-MBS),
N-succinimidyl-[4-(iodoacetyl)amino]benzoic acid ester (SIAB),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid ester (SMCC),
succinimidyl-4-(p-maleimidophenyl)butyric acid ester (SMPB),
N-succinimidyl-3-(2-pyridyldithio)propionic acid ester (SDPD),
4-[3-(2,5-dioxo-3-pyrrolinyl)propionyloxy]-3-oxo-2,5-diphenyl-2,3-dihydrothiophene-1,1-dioxide,
acetylalanylleucylalanylaminobenzyl,
acetamido-p-thioureidobenzyl.
It is also possible to utilize bonds not of the covalent type for coupling purposes wherein ionic as well as van der Waals and hydrogen bridge bonds can contribute toward the linkage in varying proportions and strengths (key and lock principle) (for example, avidin-biotin, antibody-antigen). Also inclusion compounds (host-guest) of relatively small complexes in relatively large cavities in the macromolecule are possible.
The coupling principle resides in first producing a bifunctional macromolecule by either fusing an antibody hybridoma directed against a tumor antigen with a second antibody hybridoma directed against a complex according to this invention, or linking the two antibodies chemically via a linker (e.g. in the way set forth in J. Amer. Chem. Soc. 101:3097, 1979) or binding the antibody directed against the tumor antigen to avidin (or biotin, respectively), optionally via a linker [D. J. Hnatowich et al., J. Nucl. Med. 28:1294 (1987)]. In place of the antibodies, it is also possible to employ their corresponding F(ab) or F(abxe2x80x2)2 fragments. For pharmaceutical usage, first the bifunctional macromolecule is injected which is accumulated at the target site, and then, at a time interval, the complex compound of this invention is injected [optionally bound to biotin (or avidin)] which is coupled on at the target site in vivo and there can deploy its; diagnostic or therapeutic activity. Moreover, other coupling methods can likewise be utilized, such as, for example, xe2x80x9creversible radiolabelingxe2x80x9d described in Protein Tailoring Food Med. Uses [Am. Chem. Soc. Symp. 349 (1985)].
A particularly simple method for the production of antibody conjugates or antibody fragment conjugates is available in the form of the so-called solid phase coupling procedure: The antibody is coupled to a stationary phase (e.g. an ion exchanger) located, for example, in a glass column. By successive flushing of the column with a solution suitable for generation of aldehyde groups, washing, rinsing with a solution of the functionalized complex (or ligand), washing (in case the ligand is used, rinsing is furthermore performed with a solution containing the metal salt, followed by another rinsing step) and, finally, elution of the conjugate, very high conjugate yields are obtained.
This procedure permits the automatic and continuous production of any desired quantities of conjugates.
Also other coupling steps can be performed in this way.
Thus, for example, fragment conjugates can be prepared by the sequence of papain reduction/bi-functional linker/functionalized complex or ligand.
The thus-formed compounds are subsequently purified preferably by chromatography by way of ion exchangers on a fast protein liquid chromatography unit.
The metal complexes of this invention are produced as disclosed in German Laid-Open Application 3,401,052 by dissolving or suspending the metal-oxide or a metallic salt (e.g. the nitrate, acetate, carbonate, chloride or sulfate) of the element of atomic numbers 21-29, 42, 44, 57-83 in water and/or in a lower alcohol (such as methanol, ethanol or isopropanol), and reacting with a solution or suspension of the equivalent amount of the complexing ligand and subsequently, if desired, substituting any acidic hydrogen atoms present in the acid or phenol groups by cations of inorganic and/or organic bases or amino acids.
Introduction of the desired metal ions can take place at the stage of the complexing compounds Ixe2x80x2A, Ixe2x80x2B or Ixe2x80x2C, i.e. prior to coupling to the cascade polymers, as well as after the coupling of the unmetalated ligands Ixe2x80x2A, Ixe2x80x2B or Ixe2x80x2C.
Neutralization takes place herein with the aid of inorganic bases (e.g. hydroxides, carbonates or bicarbonates) of, for example, sodium, potassium, lithium, magnesium or calcium and/or organic bases, such as, inter alia, primary, secondary and tertiary amines, e.g. ethanolamine, morpholine, glucamine, N-methyl- and N,N-dimethylglucamine, as well as basic amino acids, such as, for example, lysine, arginine and ornithine, or of amides from originally neutral or acidic amino acids.
In order to prepare the neutral complex compounds, it is possible, for example, to add to the acidic complex salts in an aqueous solution or suspension such an amount of the desired bases that the neutral point is obtained. The resultant solution can then be concentrated to dryness under vacuum. It is frequently advantageous to precipitate the thus-formed neutral salts by adding water-miscible solvents, e.g. lower alcohols (methanol, ethanol, isopropanol and others), lower ketones (acetone and others), polar ethers (tetrahydrofuran, dioxane, 1,2-dimethoxyethane and others) and to obtain in this way crystallized products which can be easily isolated and readily purified. It proved to be especially-advantageous to add the desired base as early as during the complex formation to the reaction mixture and thereby to save a process step.
In case the acidic complex compounds contain several free acidic groups, it is frequently expedient to produce neutral mixed salts containing inorganic as well as organic cations as the counterions.
This can be done, for example, by reacting the complex forming ligand in an aqueous suspension or solution with the oxide or salt of the element yielding the central ion, and with half the amount of an organic base required for neutralization; isolating the thus-formed complex salt; purifying same if desired; and then combining, for complete neutralization, with the needed amount of inorganic base. The sequence of addition of the bases can also be reversed.
Another possibility of obtaining neutral complex compounds resides in converting the remaining acid groups in the complex entirely or partially into esters or amides, for example. This can be done by subsequent reaction at the finished polymer complex (e.g. by exhaustive reaction of the free carboxy groups with dimethyl sulfate), as well as also by the use of a suitably derivatized substrate for introducing the complexing units of general Formulae Ixe2x80x2A, Ixe2x80x2B and Ixe2x80x2C (e.g. N3-(2,6-dioxomorpholinoethyl)-N6-(ethoxycarbonylmethyl)-3,6-diazaoctanedioic acid).
The conjugates of antibody and complex are dialyzed, prior to in vivo use, after incubation with a weak complexing agent, such as, for example, sodium citrate, sodium ethylenediaminetetraacetic acid, in order to remove weakly bound metal atoms.
The pharmaceutical agents of this invention are likewise produced in a manner known per se by suspending or dissolving the complex compounds of this inventionxe2x80x94optionally combined with the additives customary in galenic pharmacyxe2x80x94in an aqueous medium and then optionally sterilizing the suspension or solution. Suitable additives are, for example, physiologically acceptable buffers (such as, for instance, tromethamine), additions of complexing agents (e.g. diethylenetriaminepentaacetic acid) orxe2x80x94if requiredxe2x80x94electrolytes, e.g. sodium chloride orxe2x80x94if necessaryxe2x80x94antioxidants, such as ascorbic acid, for example.
If suspensions or solutions of the compounds of this invention in water or physiological saline solution are desirable for enteral administration or other purposes, they are mixed with one or several of the auxiliary agents (e.g. methylcellulose, lactose, mannitol) and/or tensides (e.g. lecithins, xe2x80x9cTweenxe2x80x9d, xe2x80x9cMyrjxe2x80x9d) and/or flavoring agents to improve taste (e.g. ethereal oils), as customary in galenic pharmacy.
In principle, it is also possible to produce the pharmaceutical agents of this invention without isolating the complex salts. In any event, special care must be taken to effect chelate formation so that the salts and salt solutions according to this invention are practically devoid of uncomplexed, toxically active metal ions.
This can be ensured, for example, with the aid of dye indicators, such as xylenol orange, by control titrations during the manufacturing process. Therefore, the invention also concerns processes for the production of the complex compounds and their salts. A final safety measure resides in purifying the isolated complex salt.
The pharmaceutical agents of this invention preferably contain 1 xcexcmol to 1 mol/l of the complex salt and are normally made into doses in amounts of 0.0001-5 mmol/kg. They are intended for enteral and parenteral administration. The complex compounds according to this invention are utilized
(1) for NMR and X-ray diagnostics in the form of their complexes with the ions of the elements with atomic numbers 21-29, 39, 42, 44 and 57-83;
(2) for radiodiagnostics and radiotherapy in the form of their complexes with the radioisotopes of the elements with atomic numbers 27, 29, 31, 32, 37-39, 43, 49, 62, 64, 70, 75 and 77.
The agents of this invention meet the variegated requirements for being suitable as contrast media for nuclear spin tomography. Thus, they are excellently suited for improving the informative content of the image obtained with the aid of the NMR tomograph upon oral or parenteral administration, by increasing the signal intensity. Furthermore, they exhibit the high efficacy necessary to introduce into the body a minimum amount of burdening foreign substances, and they show the good compatibility required for maintaining the noninvasive character of the examinations.
The good water solubility and low osmolality of the compounds of this invention make it possible to prepare highly concentrated solutions, thus maintaining the volume load on the circulation within tolerable limits and compensating for dilution by body fluid, i.e. NMR diagnostic aids must exhibit. 100-1,000 times the water solubility of agents for NMR spectroscopy. Furthermore, the agents of this invention exhibit not only a high in vitro stability but also a surprisingly high stability in vivo so that release or exchange of the ionsxe2x80x94actually toxicxe2x80x94not bound in a covalent fashion in the complexes takes place only extremely gradually within the time period during which the novel contrast media are again completely eliminated.
In general, the agents of this invention are used, for NMR diagnostic aids, in doses amounting to 0.0001-5 mmol/kg, preferably 0.005-0.5 mmol/kg. Details of use are discussed, for example, in H. J. Weinmann et al., Am. J. of Roentgenology 142 619 (1984).
Especially low doses (below 1 mg/kg b6dy weight) of organ-specific NMR diagnostic aids are usable, for example, for the detection of tumors and of cardiac infarction.
Furthermore, the complex compounds according to this invention can be employed with advantage as susceptibility reagents and as shift reagents for in vivo NMR spectroscopy.
The agents of this invention, based on their favorable radioactive properties and good stability of the complex compounds contained therein, are also suited as radiodiagnostic agents. Details of their usage and dosage are described, for example, in xe2x80x9cRadiotracers for Medical Applicationsxe2x80x9d, CRC Press, Boca Raton, Fla.
Another imaging method with radioisotopes is the positron emission tomography, using positron-emitting isotopes, such as, for example, 43Sc, 44Sc, 52Fe, 55Co and 68Ga (Heiss, W. D.; Phelps, M. E.: Positron Emission Tomography of Brain, Springer publishers, Berlin, Heidelberg, N.Y. 1983).
The compounds of this invention can also be utilized in radioimmuno- or radiation therapy. This process differs from the corresponding-diagnostics only in the quantity and type of isotope employed. The objective herein is the destruction of tumor cells by high-energy shortwave radiation with a minimum range. Suitable xcex2-emitting ions are, for example, 46Sc, 47Sc, 48Sc, 72Ga, 73Ga and 90Y. Suitable a-emitting ions exhibiting short half-life periods are, for example, 211Bi, 212Bi, 213Bi and 214Bi, wherein 212Bi is preferred. A suitable ion emitting photons and electrons is 158Gd which can be obtained from 157Gd by neutron capture.
If the agent of this invention is intended for use in the version of radiation therapy proposed by R. L. Mills et al. [Nature, vol. 336:787 (1988)], then the central ion must be derived from a Mxc3x6ssbauer isotope, such as, for example, 57Fe or 151Eu.
In the in vivo administration of the therapeutic agents according to this invention, they can be given together with a suitable carrier, e.g. serum or physiological sodium chloride solution and together with another protein, such as, for example, human serum albumin. The dosage herein is dependent on the type of cellular disorder, the metal ion used, and the type of imaging method.
The therapeutic media of this invention are, e.g., administered parenterally, preferably intravenously.
Details of usage of radiotherapeutic agents are discussed, for example, in R. W. Kozak et al., TIBTEC, October 1986, 262.
The agents of this invention are excellently suited as X-ray contrast media; in this connection, it is to be especially emphasized that they reveal no indication of anaphylaxis-type reactions, known from iodine-containing contrast media, in biochemical-pharmacological studies. They are particularly valuable, on account of the favorable absorption properties in regions of higher tube voltages, for digital subtraction techniques.
In general, the agents of this invention are utilized, for administration as X-ray contrast media, analogously to, for example, meglumine diatrizoate, in doses amounting to 0.1-5 mmol/kg, preferably 0.25-1 mmol/kg.
Details of utilization of X-ray contrast media are discussed, for example, in Barke, xe2x80x9cRxc3x6ntgenkontrastmittelxe2x80x9d [X-Ray Contrast Media], G. Thieme, Leipzig (1970) and P. Thurn, E. Bxc3x6cheler, xe2x80x9cEinfxc3x6hrung in die Rxc3x6ntgendiagnostikxe2x80x9d [Introduction to X-Ray Diagnostics], G. Thieme, Stuttgart, N.Y. (1977).
In summation, the synthesis has been accomplished of novel complexing compounds, metal complexes and metal complex salts, opening up new possibilities in diagnostic and therapeutic medicine. This development appears to be desirable, above all in light of the evolution of novel imaging methods in medical diagnostics.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight.
The entire disclosures of all applications, patents and publications, if any, cited above and below, and of corresponding application Federal Republic of Germany P 39 38 992.8, filed Nov. 21, 1989, are hereby incorporated by reference.